Perhaps the two most comprehensive theories explaining aging in my treatise are Programmed Epigenomic Changes and Stem Cell Supply Chain Breakdown. Recent research related to the epigenetics of stem cells deals with the profound underlying relationships between those two theories. The research relates to questions such as “What causes a stem cell to proliferate (e.g. reproduction through mitosis making more stem cells of the same kind), and what causes a stem cell to differentiate (e.g. generate more specific progenitor or somatic cells)? The subject has been called Epigenetic alchemy for cell fate conversion. I review some of that current research here. The key players I am going to focus on here are DNA methyltransferases and their key regulatory roles.
Background on DNA methylation
I have discussed DNA methylation and its role in aging in a number of my earlier blog entries. See for example Epigenetics, epigenomics and aging, DNA methylation, personalized medicine and longevity and Histone acetylase and deacetylase inhibitors, DNA demethylation – a new way of coming at cancers, and Epigenetics going mainstream.
“DNA methylation, particularly when applied to CG-rich promoter sequences, has been shown to silence gene expression in a heritable manner. DNA methylation is therefore a form of cellular memory. Because DNA methylation is not encoded in the DNA sequence itself, it is called an epigenetic modification (“epi”, Greek origin: “above” or “upon”). The transcriptional silencing associated with 5-methylcytosine is required for fundamental biological processes such as embryonic development, protection against intragenomic parasites, X-inactivation(ref).”,
It has long been known that “DNA methylation is impacted by aging and impacts on aging(ref). Methylation in the promoter region of genes is thought generally to be associated with gene silencing. Longevity-related and health-promoting genes may be turned off in the process of aging due to progressive methylation(ref).” I remind my readers that the 13th theory of aging covered in my treatise, Programmed Epigenomic Changes, envisages aging as a systematically articulated set of epigenomic changes including changes in DNA methylation in cells accumulated with aging. One researcher goes so far as to assert that DNA methylation is the cause of aging. See my blog entry Homicide by DNA methylation.
Much of the new research relates to the life-and-death roles of DNA methyltransferases in adult stem cells and what causes stability in embryonic stem cells. “– the DNA methyltransferase (DNA MTase) family of enzymes catalyze the transfer of a methyl group to DNA. DNA methylation serves a wide variety of biological functions. All the known DNA methyltransferases use S-adenosyl methionine (SAM) as the methyl donor(ref).” As mentioned, a methyl group transferred to a GpC site in the promoter region(ref) of a gene generally serves to silence that gene. “CpG sites are regions of DNA where a cytosine nucleotide occurs next to a guanine nucleotide in the linear sequence of bases along its length. “CpG” is shorthand for “—C—phosphate—G—“, that is, cytosine and guanine separated by a phosphate(ref).”
The new research relates to How DNA methyltransferases 1. initiate and maintain methyl marks, 2. are involved in self-renewal of embryonic stem (ES) cells, and 3. act in somatic (adult) stem cells including: hematopoietic, epithelial, neural and muscle cells. It also relates to the molecular factors that keep embryonic stem cells from differentiating and the role of methyltransferases once those cells start differentiating. I start out with research on adult stem cells.
DNA methyltransferases and adult stem cells
The April 2010 review publication DNA methylation in adult stem cells: New insights into self-renewal summarizes the important role of methyltransferases in preserving adult stem cell lineages. “Methylation of cytosine residues in the context of CpG dinucleotides within mammalian DNA is an epigenetic modification with profound effects on transcriptional regulation. A group of enzymes, the DNA methyltransferases (DNMTs) tightly regulate both the initiation and maintenance of these methyl marks. Loss of critical components of this enzymatic machinery results in growth, viability and differentiation defects in both mice and humans, supporting the notion that this epigenetic modification is essential for proper development. Beyond this, DNA methylation also provides a potent epigenetic mechanism for cellular memory needed to silence repetitive elements and preserve lineage specificity over repeated cell divisions throughout adulthood. Recent work highlighting the specialized roles of DNA methylation and methyltransferases in maintaining adult somatic stem cell function suggests that further dissection of these mechanisms will shed new light on the complex nature of self-renewal.”
The 2010 study DNMT1 maintains progenitor function in self-renewing somatic tissue gets down to more specifics. “Progenitor cells maintain self-renewing tissues throughout life by sustaining their capacity for proliferation while suppressing cell cycle exit and terminal differentiation. DNA methylation provides a potential epigenetic mechanism for the cellular memory needed to preserve the somatic progenitor state through repeated cell divisions. DNA methyltransferase 1 (DNMT1) maintains DNA methylation patterns after cellular replication. Although dispensable for embryonic stem cell maintenance, the role for DNMT1 in maintaining the progenitor state in constantly replenished somatic tissues, such as mammalian epidermis, is unclear. Here we show that DNMT1 is essential for epidermal progenitor cell function. DNMT1 protein was found enriched in undifferentiated cells, where it was required to retain proliferative stamina and suppress differentiation. In tissue, DNMT1 depletion led to exit from the progenitor cell compartment, premature differentiation and eventual tissue loss. Genome-wide analysis showed that a significant portion of epidermal differentiation gene promoters were methylated in self-renewing conditions but were subsequently demethylated during differentiation. — These data demonstrate that proteins involved in the dynamic regulation of DNA methylation patterns are required for progenitor maintenance and self-renewal in mammalian somatic tissue.”
The 2009 publication DNA methyltransferase 1 is essential for and uniquely regulates hematopoietic stem and progenitor cells establishes a similar critical role for DNMT1 in hematopoietic stem and progenitor cells. “DNA methylation is essential for development and in diverse biological processes. The DNA methyltransferase Dnmt1 maintains parental cell methylation patterns on daughter DNA strands in mitotic cells; however, the precise role of Dnmt1 in regulation of quiescent adult stem cells is not known. To examine the role of Dnmt1 in adult hematopoietic stem cells (HSCs), we conditionally disrupted Dnmt1 in the hematopoietic system. Defects were observed in Dnmt1-deficient HSC self-renewal, niche retention, and in the ability of Dnmt1-deficient HSCs to give rise to multilineage hematopoiesis. Loss of Dnmt1 also had specific impact on myeloid progenitor cells, causing enhanced cell cycling and inappropriate expression of mature lineage genes. Dnmt1 regulates distinct patterns of methylation and expression of discrete gene families in long-term HSCs and multipotent and lineage-restricted progenitors, suggesting that Dnmt1 differentially controls these populations. These findings establish a unique and critical role for Dnmt1 in the primitive hematopoietic compartment.”
The methyltransferases are also important in maintaining genomic stability of neural stem cells. Then 2009 study Cellular epigenetic modifications of neural stem cell differentiation reports : “Emerging information indicates that epigenetic modification (i.e., histone code and DNA methylation) may be integral to the maintenance and differentiation of neural stem cells (NSCs), but their actual involvement has not yet been illustrated. In this study, we demonstrated the dynamic nature of epigenetic marks during the differentiation of quiescent adult rat NSCs in neurospheres. A subpopulation of OCT4(+) NSCs in the neurosphere contained histone marks, trimethylated histone 3 on lysine 27 (3me-H3K27), 2me-H3K4, and acetylated H4 (Ac-H4). A major decrease of these marks was found prior to or during differentiation, and was further diminished or reprogrammed in diverse subpopulations of migrated NSCs expressing nestin or beta-III-tubulin. –. Furthermore, we found an outward translocation of DNA methylation marker 5-MeC, DNMT1, DNMT3a, and MBD1 in NSCs as differentiation began and proceeded; 5-MeC from homogeneous nucleus to peripheral nucleus, and DMNT1a and 3a from nuclear to cytoplasm, indicating chromatin remodeling. — These results indicate that chromatin is dynamically remodeled when NSCs transform from the quiescent state to active growth, and that DNA methylation modification is essential for neural stem cell differentiation.”
Embryonic and induced pluripotent stem cells and maintenance of pluripotency
The methyltransferases play somewhat of a different role when it comes to fully pluripotent cells – embryonic stem cells and, most likely, induced pluripotent stem cells. Philosophically, I like the position taken in the 2008 paper Capturing pluripotency. “In this Essay, we argue that pluripotent epiblast founder cells in the embryo and embryonic stem (ES) cells in culture represent the ground state for a mammalian cell, signified by freedom from developmental specification or epigenetic restriction and capacity for autonomous self-replication. We speculate that cell-to-cell variation may be integral to the ES cell condition, safe-guarding self-renewal while continually presenting opportunities for lineage specification.”
A key question is “When does a pluripotent stem cell like an Esc or iPSC stay pluripotent and when does it differentiate into a less-pluripotent state? Addressing this question is the September 2009 publication Regulation of stem cell pluripotency and differentiation involves a mutual regulatory circuit of the NANOG, OCT4, and SOX2 pluripotency transcription factors with polycomb repressive complexes and stem cell microRNAs. “Coordinated transcription factor networks have emerged as the master regulatory mechanisms of stem cell pluripotency and differentiation. Many stem cell-specific transcription factors, including the pluripotency transcription factors, OCT4, NANOG, and SOX2 function in combinatorial complexes to regulate the expression of loci, which are involved in embryonic stem (ES) cell pluripotency and cellular differentiation. This review will address how these pathways form a reciprocal regulatory circuit whereby the equilibrium between stem cell self-renewal, proliferation, and differentiation is in perpetual balance. We will discuss how distinct epigenetic repressive pathways involving polycomb complexes, DNA methylation, and microRNAs cooperate to reduce transcriptional noise and to prevent stochastic and aberrant induction of differentiation. We will provide a brief overview of how these networks cooperate to modulate differentiation along hematopoietic and neuronal lineages.”
Also addressing the same question is the 2008 publication Esrrb activates Oct4 transcription and sustains self-renewal and pluripotency in embryonic stem cells. “The genetic program of embryonic stem (ES) cells is orchestrated by a core of transcription factors that has OCT4, SOX2, and NANOG as master regulators. Protein levels of these core factors are tightly controlled by autoregulatory and feed-forward transcriptional mechanisms in order to prevent early differentiation. Recent studies have shown that knockdown of Esrrb (estrogen-related-receptor beta), a member of the nuclear orphan receptor family, induces differentiation of mouse ES cells cultured in the presence of leukemia inhibitory factor. – Supporting all of these data, stable transfection of Esrrb in ES cell lines proved sufficient to sustain their characteristics in the absence of leukemia-inhibitory factor. In summary, our experiments help to understand how Esrrb coordinates with Nanog and Oct4 to activate the internal machinery of ES cells.”
These two studies suggest that, unlike the case for adult stem cells, other factors like Esrrb are important in maintaining the undifferentiated status of fully pluripotent stem cells. This result was also observed in a 2006 mouse study which concluded “Our results indicate that ES cells can maintain stem cell properties and chromosomal stability in the absence of CpG methylation and CpG DNA.” The 2010 publication Polycomb complexes act redundantly to repress genomic repeats and genes also suggest other mechanisms that inhibit differentiation in ESCs.
What causes embryonic stem cells to differentiate? The 2009 report Cdk2ap1 is required for epigenetic silencing of Oct4 during murine embryonic stem cell differentiation indicates “Here, we show that Cdk2ap1, a negative regulator of Cdk2 function and cell cycle, promotes Oct4 promoter methylation during murine embryonic stem cell differentiation to down-regulate Oct4 expression.”
When ESCs do differentiate, then promoter methylation comes into play as indicated in the April 2010 publication Targeting of de novo DNA methylation throughout the Oct-4 gene regulatory region in differentiating embryonic stem cells. “Differentiation of embryonic stem (ES) cells is accompanied by silencing of the Oct-4 gene and de novo DNA methylation of its regulatory region. Previous studies have focused on the requirements for promoter region methylation. We therefore undertook to analyze the progression of DNA methylation of the approximately 2000 base pair regulatory region of Oct-4 in ES cells that are wildtype or deficient for key proteins. We find that de novo methylation is initially seeded at two discrete sites, the proximal enhancer and distal promoter, spreading later to neighboring regions, including the remainder of the promoter. De novo methyltransferases Dnmt3a and Dnmt3b cooperate in the initial targeted stage of de novo methylation. Efficient completion of the pattern requires Dnmt3a and Dnmt1, but not Dnmt3b. Methylation of the Oct-4 promoter depends on the histone H3 lysine 9 methyltransferase G9a, as shown previously, but CpG methylation throughout most of the regulatory region accumulates even in the absence of G9a.”
Summarizing the situation:
· The pluripotency and differentiation of ESCs and iPSCs are regulated by complex networks which maintain dominance of cell ground-state pluripotency transcription factors like OCT4, SOX2 and NANOG until differentiation is triggered. Apparently, the methyltransferases do not play a dominant role in that process though they may be involved.
· When ESCs start to differentiate, silencing of the OCT4 gene seems to take place via promoter methylation of this gene. At that point methyltransferases become important for maintaining lineages of adult stem cells
.· Adult stem cells, including neural progenitor cells and hematopoietic stem cells depend on DNA methylation for their survival in undifferentiated state. This methylation in turn depends critically on the actions of DNA methyltransferases. In plain language, the methyltransferases keep lineages of adult stem cells continuing in their niches throughout life instead of having all the adult cells differentiating early in life leaving no reserves of such cells.
So, DNA promoter regulation via methylation in stem cells is an important mechanism for the operation of the stem cell supply chain.
There is also a growing number of publications on DNA methylation and the role of methyltransferases in cancer stem cells, and I will probably take that topic up in a later blog post.